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Neuroimmunology - Science topic

Neuroimmunology are neuroscience, Immunology,Neuroimmunological interactions during physiological conditions and disease
Questions related to Neuroimmunology
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If you are aware of the fact that cancer patients treated with standard chemotherapy over the past 20 or so years have cognitive problems with reasoning and motor skills, I'd like your input on why, and why ten to twenty years later.  Microglia (brain macrophages, which is a misnomer) can initiate the process of neurodegeneration, which leads to ex-cancer patients, who've survived through chemotherapy, down the path of significant brain damage.  There is an initial insult due to systemic inflammation, but I'm thinking that endothealial damage and their ability to secrete inflammatory lymphokines perpetuate or exhasterbate chemotherapy-induced cognitive problems in cancer patients.
What do you think?
Michael
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Microplastics can cross the blood brain barrier and when they do they encounter the brains microglia. The microglia act as the brains immune system and attempt to swallow the microscopic fragments. This causes them to swell and then they can block blood flow and cause neuronal damage. The microplastics cause an inflammatory response which can cause behavioural disturbances. Plastics interfere with the microglia’s ability to regulate connections between neurons. This in turn reduces brain plasticity and accelerates neuronal degeneration.
Is hardly surprising that Parkinson’s Disease and Alzheimer’s Disease have increased in the last 20 years.
In brain autopsies carried out in 2024 the amount of microplastics in human brain tissue had increased by 50% compared with a sample from 2016.
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I am analyzing the immune cells in neuronal cell bodies using flow cytometry and noticed two diagonal populations in my FSC-A vs. FSC-H plot. I have excluded the dead cells.
Is it possible that both populations are singlets?
I've attached the plot below.
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Your sample most likely also contains doublets. To keep only your singlets, select the main population that goes throught the line x=y
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Hi, I'm a student who studies neuroimmunology.
I want to analyze mouse vagus nerve with RT-qPCR like this thesis but I don't know how to dissect the vagus nerve I found some protocol videos for DRG neurons from a mouse but there are not many protocols for dissecting VG, and even there are no protocol video for VG nerve dissection in Youtube. Could you recommend any good protocol for VG dissection?
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RNA isolation from mouse DRG and JG/NG (VG) and qRT-PCR
Mouse DRG or nodose ganglia (NG)/jugular ganglia (JG) (combined VG) were harvested and homogenized with a bead homogenizer (BioSpec) in lysis buffer RA1 (Macherey-Nagel). Total RNA was extracted with the NucleoSpin RNA isolation kit following manufacturer’s protocol. Equal amounts of cDNA were synthesized from total RNA extracts using the iScript cDNA Synthesis kit. Gene expression levels were determined using gene-specific primers (key resources table) and 2X qPCR Universal Green MasterMix (Lamda Biotech) or Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific). Reactions were cycled using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific) or QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) using the manufacturer’s protocol. Gene expression was normalized to Gapdh, and relative expression was calculated using the ΔΔCt method.
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It is accessible easily in the neck region. you may need a dissection lens since its pretty small in your model animals
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Hello! I've recently been trying to optimise immunofluorescent staining of mouse meningeal tissue, and I'm having trouble mounting it just because it's so thick.
We use prolong to mount and usually ~10 uL is enough for our 10 uM brain cryosections but even 20 uL seemed to be too little for the meninges since the meninges is much thicker (cover slip wouldn't rest on the tissue properly).
Would anyone have any advice on this? I feel like the process would be similar to mounting thicker cryosections of regular tissue.
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Personally I've not had to mount really thick sections, but I've seen other people make a shallow well on the slide by gluing coverslips to the slide. This might help keep the coverslip flat.
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I was wondering if anyone knows an antibody that can be used for staining microglia in zebrafish? I am familiar with 4c4 and L-plastin antibodies, but these are not perfect for me. Is anyone familair with, for example, a P2Y12 ab that works in zebrafish ?
Thanks!
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Bommana Raghunath Reddy, Anna A. Akopyan Sorry for bothering you; following this thread, I was wondering if I could ask for more details on this antibody from your experience. We recently purchased this antibody, and I've tried it on frozen zebrafish brain (10 um) but haven't seen signals with goat anti-rabbit Alexa 488. I read previous articles that showed a strong signal presentation (for example. ). I'm just curious if there is something I have been missing or if I did it wrong. I tried using citrate as antigen retrieval at 80c for 30 mins (this worked for L-plastin) and 110 c, 16 mins. Was wondering if I could have suggestions from you. Thank you.
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I always hear about some myths in neurology, and mostly about stroke but I want to know what myths other diseases have.
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Time
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A peer-reviewed journal, Neuroimmunology and Neuroinflammation opened a special issue to publish review articles about recent advances on Parkinson's disease. Please refer the link above to have more information how to submit it. Thanks.
Backil
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Thank you very much
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Hello experts,
I want to ask about CNS in the Neuroinflammation/Neuroimmunology concept.
Is it correct to say that CNS is defined as immune privilege organ due to its unique of immune reaction compare with the general immune system? Is the unique refers to the ability of CNS to induce its own immune reaction, independent from other peripheral immunity, or any other reasons?
And is it still updated to classify CNS as immune privileged system?
Thanks in advance for your answers and comments
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Fakhruddin Masse Originally thought to be immunologically privileged, now it is accepted that immune cells are present in the meninges and provide immune surveillance of the CNS. However, the unique properties of these lymphatic systems, including their location and size, may explain why immune responses to CNS antigens are often slower than in peripheral tissues.
Reference: doi: 10.1016/j.it.2015.08.006
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Hello everyone,
I was wondering if anyone knows another protocol for myelin purification other than the one published by Norton & Poduslo, 1973.
I want to do a phagocytosis assay ex vivo with primary microglia and myelin, and in vivo I want to inject it into the prefrontal cortex of mice to evaluate phagocytosis activity of the microglia from that region.
However, I'm finding a bit difficult to get access to an ultracentrifuge as described in the Norton & Poduslo method.
Any suggestions are well appreciated.
thank you all
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Hi Marcela, you might want to check Laroca and Norton, 2007- . It's slightly different with some modifications, although based on the previous.
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Hello everybody
I've been trying to seed microglia from adult mice and I haven't been successful.
I isolated them using Miltenyi Beads and that worked perfectly, passed them through the cytometer and confirmed my isolation worked good and about 85% were viable. I seeded them in 24-well plates with Poly-L-Lysine but microglia didn't make it throughout the night.
Any suggestions?
Thank you
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Hi Marcela,
we successfully isolated microglia from the brain and cultured them overnight for in vitro experiments. We also uses CD11b-Bead sorting from Milteny. We did not use however coated plates but untreated ones.
If you like have a look at our paper. In the Materials section under "Isolation, culture, and stimulation of primary microglia" you find the brief protocol.
For any more detailed questions you can also as Anne Wolf here on ResearchGate. She is the specialist.
All the best and stay healthy,
Marc
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Over the past three years, a patient with hereditary desminopathy (Thr341Pro DES mutation in the heterozygous state) has an uneven decrease in muscle strength in the hands. In the right hand, the decrease in muscular strength in the last three years is 2.1 times, and in the left one - 1.5 times. In a weaker limb, the disease progresses faster. A similar pattern is observed in the legs. The father of the patient had the same changes.
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Hi guys!
I am advancing in the empirical design of my next project about regimes of knowledge production on Adverse Drug Reactions (ADRs) and I would like to know if you have any suggestions of literature about this topic in STS, Medical Sociology of related fields.
I am interested in the perspective of the coproduction of biomedical evidence which emerges in the circuit of pathologization/diagnostic/medicalization/evaluation/new research agenda, specially addressed to the diffusion of immunotherapies and artificial antibodies in Oncology and Rheumatology.
Keep safe and cheers!
Renan
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Wow, Barry, Renan can now go wild.
Those are the most important references.
I recall the frustrations of a US doctor who penned that as:
Death by Prescription - Dr Ray D Strand
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Within three years, a patient with a desminopathy (Thr341Pro DES mutation) was found to have a 17% increase in the level of C4 complement components to 0.41 g / l (Norm 0.1-0.4 g / l).
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Dear Yosvany Castillo! Thank you very much for your answer. Over the past 2 years in this patient with desminopathy (Thr341Pro DES mutation in the heterozygous state), the C4 level of the complement component decreased to 0.36 g/l without taking medications.
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Does OLig2 ab stain all oligodendrocytes? i.e. progenitor, postmitotic and mature oligodendrocytes in embryonic , postnatal and adult brain. I mean is there a single antibody that can stain all developmental and mature oligodendrocyte (regardless the developmental stage).  
If not, I would appreciate if anyone recommend a master marker for all oligodendrocytes.
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Olig1 & Olig2 are sustaned marker of oligodendrocyte almost express in all of lineage duration, from primary to mature oligodendrocyte (also may express in some other cells). In first step of differentiation NG2 and PDGFR express in OPCs and then sox10 expression is sign for myeline formation unset and OL maturation. O4 and CNPase are marker of early mature OL and MBP, PLP and MOG are marker of fully mature OLs.
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I want to look at the expression of CD68 (ED 1)- marker of activated microglia in rat (sprague dawley) brain sections (hippocampus and medial prefronal cortex). The animals were perfused transcardially with saline followed by 4% PFA. 40um sections were then cut on a vibratome. I tried the abcam anti CD68 antibody (ab 31630) with different antigen retrieval methods (boiling, boiling+citrate and boiling+Tris) but could not detect any staining. I had also stained the sections simultaneously for Iba-1, a general marker for microglia which worked excellently. I want to know:
1) If anyone knows of a CD68 antibody which works well in rat brain sections and the protocol for the IHC
2) If anyone has successfully been able to detect CD68 expression using the abcam antibody. If yes, then kindly share the protocol.
Thanks! 
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I used Serotec as well and worked perfectly well as long as you induce CD68 expression with pro-inflammatory agents, such as LPS. However, if you want to assess its expression in physiological conditions there is no difference from the background...
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I want to study the demyelination degree of EAE mice. I know that in the EAE model, lesions are preferentially located in the spinal cord and due to that it is very common to use LFB staining for spinal cord. Nevertheless I´m also interested in analyzing the brain. Has anyone used this staining in brain frozen tissue?
Thank you very much in advice
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Hi Inaki,
LFB may be to colorful to visualize the fiber or demyelination. Better to use silver staining if there is more demylination as it may be less sensitive if there is suttle demylination.
Following article might help you
thanks
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Basically, I am comparing gene expression in different neuroinflammatory pathways using mRNA Seq in a transgenic (KO) mouse model. The main goal is too see what genes are compensating during a neuroinflammatroy response.
What other experiments often accompany an RNA seq focused project? Western Blot? IHC? qPCR validation? And what would the purpose of those experiments serve in further elucidating the role of this missing gene and the henceforth compensatory mechanisms?
This is for my Masters thesis project so any advice would be helpful!
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Ideally both PCR and WB, but if you have budget for only one the latter makes more sense especially if you plan on publishing. For PCR validation, you could randomly choose several genes that are not differentially expressed and some genes that are differentially expressed. Genes of interested could be in the pathways that are pertinent to your investigation i.e. inflammation. I will give you an example of how my previous PI represented microarray results to give you an idea.
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i can not find any foundations supporting cancer immune therapy and cancer neuroimmunology research in egypt or outside. and if there how can i contact with them?
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Dr Khan,
Please read this article and it will answer all your previous qeustions
The article:Neurobiology of cancer: Interactions between nervous, endocrine
and immune systems as a base for monitoring and modulating
the tumorigenesis by the brain,seminars in cancer biology
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I have been recently trying to isolate primary microglia from mice pups brain (P0-P1). To prepare mixed microglia culture, I used 3 brains for a 75 cm2 flask. After around 10-12 days, astrocyte confluent layer is formed, and many microglia appears. I isolated those microglia by a shaking method (100-120 rpm/ 2 hrs).
At first harvest, I usually get 3-5 x 105 cells per flask. But, after the first harvest, I do not see the growth of new microglia. I only see the microglia that did not detach during the first harvest. Nevertheless, I tried to isolate for second harvest (after 1-2 weeks from 1st harvest), but end up with very low yield (2 x 104 cells).
Is there any ways to increase the yield?
Medium used: DMEM (10% FBS, 1% penicillin/streptomycin)
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I agree with Clarissa's suggestion. M-CSF (10ng/ml) is very important for microglia proliferation. We did not find the many difference between the 1st and other harvest by using the marker labeling and patch clamp.
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Over the past four years, in a patient with desminopathy (Thr341Pro DES mutation in the heterozygous state), the majority of antioxidant status indicators increased 1.2-2.0 times. Including glutathione in the blood increased 1.8 times to a value of 896 μmol / l (the norm of 500 - 1500 μmol / l), and the level of coenzyme Q10 in the blood increased 1.8 times to a value of 0.8 mg / l (norm 0,4 - 1,6 mg / l), vitamins E and C increased by 1.3 times, respectively, to 6.4 and 6.2 μg / ml.
In the literature there are conflicting data on their effect on the human body.
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Dear Muneeb and Anne, many thanks for your answers and recommendations.
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Our neuroimmunology lab currently has a BD Accuri C6 that has broken about every year and a half since it’s purchase (about 5 yrs old).  Valve issues, fluidics leaking onto electronics, flow cell issues, laser problems, etc.  We have about 50k to spend on a new flow cytometer.  BD has said they’ve fixed these issues, but I am hesitant.  We only need the two lasers and 4 color system.  We mostly stain immune cells with FITC, PE, PerCPCy5.5 (or 7AAD), and APC panels.  It’s used about every other day for an hour or two.  I’ve been researching the NovoCyte, Guava easyCyte, and CytoFLEX too, but my PI wants to stick with the familiar (plus they are running a great deal at about 38k with 3 years warranty).  Anyone have experience with the new BD Accuri C6 Plus? 
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Dear, Andor,
we have figured it out of how to solve the problems with BD Accuri C6 flow cytometer.
1) disassemble the machine and check the valves for cloging,
2) usually it is possible to clean the valves and keep them running if you don't use FBS, or other very sticky mediums in the future. Otherwise valves will clog again,
3) in case to have additional valves buy them from Accuri. They have designed new valves according to this problem and sell them ~460 $ each, or around ~2000 $ all the chip with all 5 valves,
4) there is another choice, to buy equivalent valves from The Lee Company and insert them in place of old clogged ones.
In general, it was terribly exhausting and money wasting years trying to understand and fix the machine but it works.
If there are people in a process of deciding whether to buy Accuri and save, or to buy other, possibly better but more expensive aparatus, don't save.
With respect,
Baltramiejus
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Over the past six months, a patient with hereditary desminopathy (Thr341Pro DES mutation in the heterozygous state) has a 1.6-fold increase in the level of the pro-inflammatory cytokine interleukin-6 to a value of 8.77 pg / ml (<7.00 pg / ml).
In addition, several parameters indicate the presence of inflammation: an increase in the number of immunity cells with markers CD25 +, CD95 +, HLA-DR + in the T-cell link; as well as an elevated level of C4 complement components.
The level of other cytokines Il-1β, Il-8, Il-10, TNF-α is normal. At the same time, the immunoregulatory index fell below the norm and is 1.19.
Details are indicated in the questions of this project "myofibrillar myopathy".
Dear scientists, please, join the discussion on this issue, do not limit yourself to viewing only. Ask me additional questions, send messages to your personal mail. Ready to answer any questions and listen to wishes.
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I am not very relative, but to the best of my knowledge IL-10 is the best antidote, similarly HemeOxygenase 1 can also be considered.
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Some tips, protocols, etc will be gratefully accepted for non paraffinized tissues but cryosections of spinal cord of 18um thickness. 
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Dear every one,
I have a proplem when i want to quantify myelin after i stained with MBP marker or Luxol fast blue. Please show me how to quantify MBP marker or Luxol fast blue?
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I am planning to design and synthesis of novel small inhibitors/antagonist of TLRs.
How should I begin? I have requested many private companies to provide samples but they could'nt provide as per their policies. So please help in this regard.
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Thank you all for your insightful comments. As suggested, I have collaborated with chemistry experts for the same.
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The chemistry of the Kit is based on two classical chemical reactions: Griess and Saville. 
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Citrate and EDTA give least free hemoglobin, heparin may interfere with the diazo-reaction.
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Is there any data comparing cyclophosphamide and MMF in acute neurolupus?
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if Presumed immunemediated is suspected you should start GCs + AZA (if mild/moderate) or CYC (if severe) or RTX (if refractory) , but if Presumed thrombotic Process is suspected (aPL abs) then you should go with full anticoagulation with IV or SC heparin followed by oral warfarin ± aspirin daily for life
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how much Brdu should be injected in mice to address smooth muscle proliferation like DSS induced Colitis?
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Thank you
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I am attempting to label proliferative cells in the rat brain (Hippocampus) using BrDU intraperitoneally. However, we are able to detect BrDU in gut cells, but not in the brain. Does anyone know why?
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My previous experience with BrdU was to study hippocampal neurogenesis, I treated mice BID 50mg/kg IP. I also did antigen retrieval 2N HCI at 37oC for 30 min followed by 0.1M Boric Acid pH 8.5 at room Temp for 10 minutes prior to blocking and primary. 
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I know there was debate as to weither once th2 always th2 while some thought there could be switching between th1 side and th2 side, while looking at profiles with GWI/ME/CFS some showed th1/th17 while others showed th2 side. I'm WONDERING IF WE MIGHT SWITCH BACK AND FORTH, DEPENDING ON WHAT MAY BE GOING ON AT THE TIME, MAYBE TH1 POINTING TO EXPOSURE OR RE-EXPOSURE TO A TRIGGER WHILE TH2 MAY POINT AT ACTIVE INFECTION WEITHER IT'S A REACTIVATED INFECTION OR A NEW ONE.
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but, it could be that while that may be the case during initial exposure, it might not be afterwards, the question now is : is it the brain controlling the immune system or is the immune system broke down. pretty sure both got damaged and with a environmental exposure basicly it's the immune system not being able to deal with a bad exposure that leads to immune system faultering and damage being done and infection following witch basicly turns into sepsis when it goes systemic and along with sinus/brain involvement the brain can receive considerable damage and meningitis makes it all a lot worse
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I want to contruct a primer for assay of scavanger receptor marker MARCO , or CD163 for rat animal model. So my question is that how to consttruct a primer for this ?
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so are we talking PCR based detection? If that is the case check out taqman probe availability for this receptor. I am pretty sure they provide kits where you can design whatcha need.
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Does anyone works with this molecule in ratt tissue? I am trying to find the best commercial antibody in order to detect CB1 receptor by immunofluoresce and western blot. 
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Hello,
I would recommend a free antibody database available at labome.com. For anti-rat CB1 antibodies for IF or WB you can check the following link: https://www.labome.com/review/gene/human/CNR1-antibody.html. Invitrogen has rabbit polyclonal CB1 antibody (ABR, PA1-743) which can be used in immunohistochemistry and WB  on rat samples (ref: López-Gallardo et al. J Neuroendocrinol. 2015). Also, Abcam rabbit polyclonal CNR1 antibody (Abcam, ab23703) was used in immunohistochemistry on rat samples at 1:300 (Meng et al, Int J Clin Exp Pathol. 2014). You can see the description of these antibodies and references following the provided links.
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Hello everybody,
I am searching for Anti-Bradykinin Receptor B2 Antibody and the phosphorylated one. I will be using brain sections from Guinea pigs and do the  immunofluorescence stain.
Note: This is the first time for me to work with Guinea pigs, and its difficult to find antibodies compatible with it.
Thank You in advance.
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Dear Amira,
 I really appreciate your answer. I was struggling with this for one month.
Thank you 
Dear Jan, 
 I'll use a rat brain sections as positive control and I'll update you with the results. Thank you 
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Hello,
I am trying to optimize our Olig2 immunohistochemistry staining protocol for PFA fixed mouse brain. I found a very helpful website that reviewed several recent publications and found people used concentrations from 1:100 to 1:500 on mouse brain, and I am planning to try several of them (https://www.labome.com/review/gene/mouse/Olig2-antibody.html) . However, on the abcam website they advise to perform heat mediated antigen retrieval with citrate buffer pH 6 for this antibody. I was wondering if anyone tried antigen retrieval for Olig2 IHC?
Otherwise, if you have experience with antigen retrieval using a microwave, could you please advice the steps? I found a protocol suggesting placing tissue sections on slides, in citrate buffer (pH 6), and boiling for 20 minutes, whereas another protocol suggested boiling for 7 minutes.
I was also wondering, after antigen retrieval, what is the best way to continue the staining protocol having the sections already on slides: i.e. the incubation with the primary antibody over night etc? I have only ever stained with the tissue inside well plates, not on slides.
Thank you,
Carola
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For microwave antigen retrieval, I use a glass Coplin jar. Put your slides in, fill with citrate buffer, no lid. Microwave on 100% power until just starts to boil; ~1 min or so. Then reduce power to 30% for 10 min. Keep an eye on it. You don't want a rapid boil. If you need, add more citrate buffer. After boil, take them out and cool on bench for about 30 min. Wash with buffer a few times then go forward as normal. I use a PAP pen to draw a circle around my tissue and then block and stain within that circle. Block 1 hr with PBS + 3% BSA and maybe a little rabbit or goat serum if you have it, then O/N 4o with primary. Next day wash and then secondary Abs for 1 hr RT. Wash and go forward with whatever method of revealing you are using, i.e. fluorescent or DAB, etc. Hope this helps.
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Does anyone know the brain concentration or % of antibody that was able to pass the BBB in patients treated with Abeta antibodies bapineuzumab or solanezumab? Thank you. 
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The papers I saw explained about 1% for general antibodies. With the dysfunction of the BBB, it may go up a few %. But remember that Abeta is in decent levels in the blood and these antibodies will likely bind targets in the blood first...
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My antibody didn't work at all. I have mouse brains, 30 um cryosectioned, and tried different concentrations of tween, as well as both formamide and SSC treatment to no avail.
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Hi Tatyana, I'm assuming you are working with 4% paraformaldehyde perfused tissue?  If so, I have had good luck with a rabbit anti-Ki67 antibody from vector labs in a free floating protocol for mouse tissue.  You can find the protocol in the paper I'm attaching.  The protocol is under "immunohistochemistry" and some images of the resulting stain are attached separately.  Hope this is helpful!
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I need to label free-floating coronal mouse brain sections. Lamentably the typical SC-52 antibody of Santa Cruz Biotechnology has been discontinued. They suggested me to try with c-Fos (E-8): sc-166940 antibody but there are not many references about it.
Thanks very much.
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I make an effort to avoid Santa Cruz's antibodies for both professional and personal reasons.  I've used c-Fos from abcam (ab209794) for immunofluorence (in mice) and it works very well.
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Dear researchgaters,
I have a question regarding staining protocols for oxidative tissue markers via IHC (4-HNE, MDA-2, 8-0HdG etc). I have found that these markers work in cryo stored material, but I have not had any success getting them to work on paraffin material. When staining with cryo, due to the poor freezing method of the brain tissue, I often have poor quality stains, high background and poor morphology on some many aspects of  cells or tissue. As the tissue for paraffin is superb in terms of quality and structure, it would be ideal to get these markers to work on this tissue.
For cryo staining I:
Acetone fix 10 min, air dry 10 min, PBS soak 10 minutes, antibody block (10%fcs + TBS or Dako) for 1/2 h -> Primary antibody overnight at 4c.
Some protocols I have seen where people stain parrafin material they are not using a retrieve step but are using .1% triton in their blocks. I have tried this during EDTA retrieve but no luck. I have tried Citrate/EDTA retrieve, and also proteinase K. Using the EDTA/Citrate retrieve I get very beautiful staining of p22phox, iNOS, SOD2, SOD1 on the parrafin embedded tissue. Can someone please help me out here! 
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Hi Jordan, put a piece of brain  (1 x 1 x1 cm in size) in ice cold 4%PFA or 10 % Formalin, what you'llget in your lab. Lieve it in the fixativ  a couple of days in the fridge. While the tissue is thawing the formalin fixation starts. After the fixation process the brain tissue as Jalpa recommaded it. The H&E stain will help you to get an impression of the quality of the tissue. Positiv controls for immunohistichemistry  will help to find out weather the reason for the negativ result is the tissue processing, the IHC protocol or the antibody by itself. To establish new antibodies or new protocols under new circumstances it is also helpfull to run a delution raw to optimize the antybody concentration. Good luck!
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Hello!
I am having trouble deciphering between fluorescent-tagged latex beads and E.coli particles that are just bound to the macrophages cell surface vs.  engulfed.
Has anyone had success washing off surface-bound latex beads or bacteria?
I am currently growing my cells on coverslips, incubating with the beads/e. coli, followed by washing with PBS 3X. However, there are still many beads bound to the glass coverslip after washing. I would like to isolate only the engulfed beads!
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It's quite difficult to wash away the extracellular particles, because they will often be bound quite tightly to multiple receptors on the cell surface. If you just need to differentiate between intracellular and extracellular particles, then there are two general approaches:
  1. Quench the extracellular fluorescence - trypan blue is often used here.
  2. Counterstain the extracellular particles with a specific reagent bearing another fluor - for example if you have antibody-coated latex beads then you can use an anti-Ig antibody for counterstaining.
In both cases you let the phagocytosis happen, and then cool the cells down and add azide to stop further phagocytosis, before quenching/counterstaining.
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CD3, CD5 Parrafin section immunohistology.
Dear Colleagues, I have also problem in Immunostaining of Parrafin sections of Rat sciatic nerve and spleen with CD3 and CD5. I am also using rabbit anti-human CD3e, #A0452, Dako as primary antibody and using Heat method for antigen retrival, but still i am unable to establish T cell staining. Please can any body gives some suggestion to improvise my stainings. Thanks
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I have used the Dako CD3 polyclonal on many animal tissues including rat and it works very well. As I recall I used it at a dilution of 1:100. The polyclonal was raised against a peptide that is very heavily conserved between species so works well in animals. Try using the Dako or novocastra polymer based secondary antibody for detection. I have stained rat speed using this antibody and got very good staining. I used both citrate and edta based antigen retrieval based solutions and either will work well. Try 5mins in a microwave or pressure cooker. Use the protein block that comes with the polymer detection kit. Straightforward HRP conjugated anti rabbit should work well enough though.  
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I have already perfused rat and fixed the brain and contusion spinal cord tissue with 4% PFA . Gonna use them for immunohistology. But now I don't have time to dehydrate and embed them into paraffin blocks immediately. I would like to know in which buffer it is convenient to leave the samples, if I want to include them in paraffin about a month (for example, ethanol, PB) or for a long time?
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If you are going to do IHC you do not want to leave them in buffer for too long. This can increase crosslinking and make the procedure more difficult to do. Not to mention extracting cytosol. I would dehydrate to 70% ethanol and proceed to the embedding at the earliest time that you could.
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I am doing double immunohistochemistry staining of rat brain sections. Unlike most protocols, I work with thicker sections (40μm, it still works very well) with all incubations done in a tissue culture plate. I follow the same protocol every time, I mix the sections during all incubation steps and still I get different staining intensities every time! Sometimes even the two hemispheres of a single section are coloured differently! I tried to optimize the conditions in multiple ways - I tried 12-well, instead of 24-well plate, with higher volumes, more mixing, extended incubation times, less sections per well, etc., yet this doesn't improve enough, as I expected. I still can't achieve regular staining and more importantly - easily comparable between experiments! Even the final step - the substrate reaction - develops differently every time! Also, the background level is hard to assess. I guess I'm missing something, I will be glad if someone with more experience can give me a clue, or has encountered similar issues, to share some suggestions on how to improve.
I am staining for c-Fos and calbindin, biotinylated secondary antibodies + streptavidin-HRP. I use two different substrates - standard DAB and a two-component Vector SG peroxidase substrate kit. All concentrations should even be in surplus. Tissue preparation has been performed similarly from the start.
Thank you!
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Hi Ivaylo,
for the free floating reactions I recommand to use the 24 well plates. Use 1 section/well and 500µl solution/well. If you like to react several sections simultaniously   take 6 well plates and 2 ml solutions/well. You can easily react more then 3 sections/well. Put he plates on an rocking-table so that the sections continiously moved and rinsed. some importenant blocking steps are necessary when you are working with Strpavidin and DAB, because this steps are very often responsible for unspecific backgroundstaining. 1. you have to block the endogenious peroxidase with H2O2 (30% H2O2 1:100 in PBS). 2. unspecific binding of secondary ab (use 10 % normal serum form the host animal of sec. ab; Goat-Anti-rabbit ==> normal Goat-serum). and 3. blocking of endogenious biotin with the Avidin/Biotin-blocking kit  (Vector Labs). Use 0.2% Triton-X100 in PBS or TBS with 1 normal-Serum as delution medium for your antibodies. Make a delution raw of your primary ab to optimize the delution and run a negativ control (no primary ab) to get an impression which part of your reaction protocol is responsible for  unspecific backgroundstaining.
Good luck!
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I would like to perform immunoprecipitation with an anti-v5 antibody followed by a mass spect analyses, on brain extract tissue. 
Where may I find the most suitable protocol ? ( anti-v5 references, which beads or which buffer should I use, and which quality control I should do )? Many thanks in advance for your answers :).
Marie.
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Hi Awatif !
Thank you for sharing ! The problem is that I can not see anymore the articles you put in attachement. Could you add it again please ?
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I am trying to use different antibodies working for trasmission electron microscope (TEM) to mark satellite glial cells (SGCs) in mice Dorsal Root Ganglia (DRGs) but none seems to work. Does anybody ever try it?
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Thank you very much for your precious advices!
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I did couple of stainings with Egr-1 in mice and I always end up with a lot of background which makes it very difficult to differentiate the positive nuclei in the tissue. I would appreciate any experience with this 
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Thanks for the response. If you have used maybe Egr-1 or c-fos for IHC from another company that has worked for you, any information regarding this, would be helpful.
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Hello. Could anyone, please, help me? I have made immunofluorescent staining ща 3 DIV primary neuronal culture made from P1 mice hippocampus. How should the beta-3 Tubulin looks like on 3 DIV? I have attached an image - can it looks like this or this is a nonsense? 
Thank you.
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Hi Nataliia,
Your astrocytes look good but your neural marker(Tub) seems distributed in a very bizarre way. Maybe you are not at the right focus or that it is unspecific staining. Try a higher magnification and show us pls your images stacked and the bright field image. I have attched an image. It should look like this.
Houda
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I have read papers highlighting ZO-1's sensitivity to overfixation, and where best results were obtained with thinner sections (10 to 20 microns). Going forward I would like to try this, but for the time being the tissue I have to work with is 50 microns thick and fixed in 4% PFA. With that said, could someone offer suggestions on how to achieve good staining under these conditions? Thank you for your time.
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Hej William, do you have a link to the paper you mention?
Hej. Florie, I'm envious of your colleague's stain. I'll try the PBS and post-fixation with actone next time. 
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I am staining for intracellular pSTAT1 and I am repeatedly running into difficulty owing to the fact I have to permeabilise with methanol (for sufficient nuclear entry of anti-pSTAT1 antibody and to facilitate STAT dimer separation).
The methanol is wreaking havoc with certain fluorochromes/antigens and causing either no successful staining, or false/bizarre stains. The problem is that it is proving almost random as to which fluorochromes/antigens are sensitive and which are resistant, for example a certain fluorochrome will work for one particular antigen and not another, and vice versa, suggesting it's a combination of both.
As of yet, I haven't found a reliable source of information to guide the design of my experiments around this, and was wondering if anyone had any ideas or know of any resource that may help me!
Thanks in advance!
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On page 8 of this document, it lists some of the methanol-resistant and methanol-sensitive fluorochromes. It isn't too extensive, but it's helped me on more than one occasion.
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Hi all!
Can a mouse whole vertebral column be stored in 4% PFA for longer than 24 hours, but shorter than one week?
I plan to put the vertebral columns of my mice in PFA on a Friday and do sucrose the following reagents on Monday. Will this affect the quality of the extracted spinal cord later on?
Thanks in advance for your help.
Cheers!
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Hi Alaa,
In my opinion you can store the tissue in 4%PFA for 48 or 72h. 
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Hi everybody,
I need a protocol and buffers to extract proteins from mouse hippocampus samples for ELISA measurement. The protein that I am going to measure is NR2B-NMDA. Samples are freeze in -80 degrees celcius.  
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I use a lisis buffer recomended for Elisa, that is
1-  5-10 mg of tissue in 50 ul of lysis buffer (Bio-Rad. Bio-Plex Cell Lysis Kit # 171-304011)
another option is use PBS buffer with protease inhibitor (HALT PROTEASE INHIBITOR COCKTAIL ref:87786, Thermo Scientific Pierce)
In both cases I sonicated the samples for 3 x 5 seg
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I would like to detect motor neurons in mouse spinal cord as an alternative to violet crystal.
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Hello,
we use an antibody against Choline-Acetyltransferase  from Millipore developed in goat (A-ChAT, gt, Millipore) and for immunofluorescence we dilute it  1:400 when afterwards detected with secondary antibodies conjugated with red fluorescent CY3 (Anti-goat FaB-Fragments developed in mouse  from Jackson).
In DAB stainings we use a dilution from 1:1000, followed by detection with a Vector-Kit.
However, if Dr. Schmidt provides you a free test sample, it is worth to test it!!
Sincerely Yours
Stefan
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I need protocol for FACS analysis of Rat Sciatic nerve. 
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I see. I suggest  you can try collagenase and dispase to digest the tissue, or papain or trypsin. After digestion you can try to triturate with glas pipette to have a single cell solution. 
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When he gives extract orally for 14 days and challenged by lps once, and performed novel object recognition. When he analysed his data,  results was not dose dependent 100  and 200 mg is dose dependent but they declined in 400 mg but toxicity studies says (literature) LD may be up to 2000 mg. So what are all the possible reasons for this case.
Please share your suggestions
Thanks in advance
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what do you mean by "extract" and "challenged by lps" ? I don't understand what you are asking.
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I'll test for neuronal autoantibodies
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Not needed but prolonged storage (years) is not recommended as it decreases reactivity of Igs. Most IgG serum  subtypes are pretty stable except for IgG3 which quickly degrades with time. To confirm, you may do ELISA at particular dilution (and same experimental conditions) of the same serum once immediately and later, after years of storage at -80 degree C. If you compare two data, you will see substantial reduction in titer of any IgG/IgM subtypes in old serum. 
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obviously theres a group of CFS'ERS AND ME/CFS'ERS that didn't get ill from viruses but instead became seceptable to them and re-activation of them and some were made ill by chemical exposures, now MCS and the sinus,olfactory,brain pathway, I think shows a whole other impact to the brain besides the stomach/bowel route. GWI has been reconized as TBI/PTSD, YET THERES STILL SO LITTLE POINTING TO THE SEVERITY OF BRAIN DAMAGE that turned my world upside down when I too fit into this box , I cant tell when my brain is affected by either route and no not all ME/CFS'ERS have the sinus/brain involvement witch is looking to not only be related to chronic rhinosinusitis but very possably severe Fibromyalgia.  I know we are getting there but gee, kind of would like to see it happen before I croak! obviously there are some in the know out there yet it seems limited to GWI research, very thankful for that but hey some of us are not GWV'S yet suffer from the same cause "chemical exposure" there's a few new articles out on MCS , one that addresses the hearing dysfunctions, can we get more articles out there that point to chemical exposures  and the tipping point (what I call it) where mast cells may suppress the immune system when they cant keep up and this allows  mucosal and tissue damage and infection to acure and not only play a role in autoimmunity but probably also the B cell switching to IgE. I can relate to both GWI and Autism ,have tried to keep up with research on both.
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désolée mais je n'ai pas de réponse
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None
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Dear Gerard,
TH is the standard as many other people has already suggested to you. However is a very early marker and sometimes you have no idea about how many mature dopaminergic neurons you have in your culture. This is a crucial point when you try to differentiate your iPS into functional dopaminergic neurons
So I suggest you to double-label your cells with TH in combination with DAT or AADC, that are late markers. Millipore sales very good antibodies for dopaminergic characterization.
Good luck!
Federica
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none
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For confocal election microscopy, we used co-localization of 2 molecular probes, but the same 2 colors green and red for better imagining.
I think magnetic beads would be another good idea. All the best!!
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Dear all, may I know if microglia cells produce leukotrienes in any form?
Thank you.
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 Hi, thanks for your suggestion. I most probably will test the supernatant with a commercial ELISA kit. 
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Looking for studies in adolescent monkeys regarding development of neuroimmune mechanisms (e.g.,microglia) or even peripheral immune mechanisms (e.g., phagocytosis, lymphocyte numbers). I'm coming up empty--any leads?
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I injected a drug (subcutaneous in mice) and I noticed that there was an increase in number of microgias in the hippocampus (IBA positive cells). These microglias are more branched and non-reactive to MHC-II.
How should I interpret this increased in the number of branches and increased in the number of not activated microglia caused by a drug compared to control animals (which did not suffer intervention)? It can be interpreted as neuroprotection phenotype even if they have not suffered any inflammatory stimulus? Is it possible that microglia become more "resting" than those of the control animals that do not have inflammatory stimulus?
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There is also an excellent new paper out from Marie Eve Tremblay showing a new type of microglia (called "dark" microglia based on their EM appearance) that becomes abundant during pathological states such as chronic stress, aging, Alzheimer's, etc. They display signs of oxidative stress, but also have highly ramified, thin processes, so it sounds like they may be relevant for your question. See paper below:
Glia. 2016 May;64(5):826-39. doi: 10.1002/glia.22966. Epub 2016 Feb 5.
Dark microglia: A new phenotype predominantly associated with pathological states.
Bisht K1, Sharma KP1, Lecours C1, Gabriela Sánchez M1, El Hajj H1, Milior G2, Olmos-Alonso A3, Gómez-Nicola D3, Luheshi G4, Vallières L1, Branchi I5, Maggi L2, Limatola C2, Butovsky O6, Tremblay MÈ1.
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  1. How long would you expect auto-antibodies to be present in CSF in autoimmune encephalitis?
  2. Would there be any difference between untreated and treated (IVIG/rituximab) cases?
  3. Could you perform a lumbar puncture several years later in untreated patients and still be able to detect the auto-antibody (AMPAR 1/2, NMDAR, GABA-bR, VGCC, CASPR-2, VGKC)?
Thank you for your time and help!
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Several aspects have to be kept in mind when discussing the persistence of auto-antibodies in the CSF. In the case of paraneoplastic syndromes the source of autoantibodies are presumably plasma cells in secondary or tertiary lymphoid structures outside the CNS. Therefore, levels of antibodies in the CSF should be linked to serum levels. It has to be noted that paraneoplastic antibodies can persist for years, even when the tumor has been removed (Alexopoulos H  et al. Paraneoplastic anti-NMDAR encephalitis: long term follow-up reveals persistent serum antibodies. J Neurol. 2011 Aug;258(8):1568-70).
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I need to fix my animals with 4% Para to do IHC on the sections later. But I also need to determine the integrity of the BBB and I have chosen the IV HRP method. I have read that most others use Karnovsky's Fixative (2% Paraformaldehyde, 2.5% Glutaraldehyde and 0.1M Buffer). Any suggestions? What are the benefits to using the Karnovsky's Fixative?
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Short rule: fixation for electron microscopy needs glutaraldehyde, without or in cobination with paraformaldehyde (concentration of each substance determines quality for the type of tissue).
Light microscopy needs only formalin or paraformaldehyde. Colouration determines other type of fixatives, e.g. alcohol based. By the way Muss's answer is correct.
Best wishes and succes. Enrico Marani
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Last year a link between a form of fetal brain damage and the mosquito-born Zika virus has been confirmed by Brazilian health authorities.
The virus, endemic in parts Africa, South America, Southeast Asia and some Pacific Islands, has until now been blamed for symptoms such as fever, mild headache, skin rashes, joint pain and conjunctivitis, or "red eye."
Initial analysis shows that the virus can be passed to a fetus and that the fetus is at greatest risk from the virus during the first three months of pregnancy.
A surge in recent months of babies born with microcephaly, or an unnaturally small brain, in Brazil's northeast, led authorities to suspect the virus may have more sinister effects than previously recorded.
Microcephalic children can suffer developmental and intellectual difficulties that limit intelligence and muscle coordination for life.
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Yes unfortunately increasingly it is looking like a Media-Induced Zika Emergency (MIZE) tied to possibly billions of dollars. Zika may well be a terrible virus causing microencephaly, but the scientific evidence to back that claim is next to nothing. Only six cases of possible Zika involvement, along with many other factors viruses/bacteria not ruled out. Really, only in six cases of microcephalie, Zika virus been found in the victims, but are the other potentials ruled out? No. As long as you have backing from MIZE, smaller/independent media and even many scientistis are getting sucked into a story that is no further than a story. If MIZE turns out to be wrong/fake, there will be noone to blame, because then they will say we are not scientists and we are only reporters. But there is potential to make lots and lots of money of this story.
best wishes, Refik
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I am trying obtain primary microglial cells from adult mice. I am using magnetic bead separation and from FACS analysis my cultures are pretty pure (around 90%) but every time I plate the cells they die!
I am using DMEM/F12 + Glutamax media and coating them onto poly-L-lysine plates. There are one or two cells that are alive, and look like microglia in each field of view but the majority are small, round and live/dead stain showed they were positive for PI.
I want to try it again but I am not sure why they are dying and what I should change next time.
Any advice/help would be great appreciated!!
Thanks,
Sam
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Hi Matthias, thanks for your reply. I have tried leaving the cells up to 5 days after culture and although a few cells appear healthy the majority just seem to die. No I haven't tried adding M-CSF yet, do you think the lack of it would cause the cells to die after isolation? How much do you add to your media?
Thanks,
Sam
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Hello everyone! I would like to find a lab host to participate to TALENTs grant. It is a ìn european grant that provide salary for 18 months, divided in 12 to spent in an Host institution and 6 to return to my base institution. I'm looking for an host laboratory situated in one of following country: Slovenia*, Croatia, Bosnia and Herzegovina, Serbia, Montenegro, Albania, Greece, Germany, France, Austria, Switzerland, Liechtenstein. The laboratory, given my CV, should ideally work in neuroinflammation / neurodegenerative diseases or I would like to learn CRISPR/CAS9 system...details in the link attached..
thanks for all the possible answers! 
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Dear Fulvio.
Sorry, I asked you, is it other program in EU or the first, you got grant ?After that you are looking for placement?
Thanks
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We want to culture hippocampal slices and we want to selectively deplete astrocytes and oligodendrocytes without affecting the neurons. We know how to deplete microglial cells and it works more or less effectively. Is there any compound (like clodronate) to deplete these cells pharmacological? We found some papers describing methods but I don't think that they are applicable in our experiments. Thanks for your answers =)!
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In addition to fluorocitrate you can try another gliotoxin: amino adipic acid. It damages astrocytes, and possiblly also oligodendrocytes. See: Aminoadipic acid toxic effects on retinal glial cells. Ishikawa Y, Mine S. Jpn J Ophthalmol. 1983;27(1):107-18
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Hi. I used mixed glial culture, then shake the primary microglia off and replate them in 96 well plate for functional assay. I saw different morphology between WT cell and gene A knockout cell. I enclosed here the CD11b staining picture of both cell types. The WT cell more look like the ramified shape, and the gene A KO cell looks more like an amoeba shape. What is the normal morphology of primary microglia from mixed glial culture? Does the gene A KO cell more like activated microglia?
Thanks!
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Update: I've just succeed to download your pictures. At first glance, your KO microglia seem to have different morphology. In particular, round flattened cells seem to be more frequent. I usually see this "amoeboid" morphotype in my cells after some pro-inflammatory stimulation connected to M1 microglia polarization, see reference attached.
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I am trying to detect cytokines TNF and IL1B in primary rat glia culture media (high cofluency, no phenol red) after 40 min treatment with amyloid b oligomers. Unfortunately I could never reach detection limit (15 pg/ml). I could also not detect anything in lysed cultures (hypotonic buffer) and after 10x concentration of cell culture media. I would be very grateful if somebody could share their experience in ELISA-based detection of cytokines in cell culture media.
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Hi Katarzyna,
I usually measure cytokine responses in cell cultures like PBMCs, T cells, macrophages, but not in glial cells. However, from your question I wonder if your stimulation experiments with oligomers have any positive controls (for example, LPS stimulation)? If so, did they also not show any detectable cytokine levels?
That would help you find out if your cultures are able to respond to any stimuli. From there on, you can continue troubleshooting your experimental and detection systems. Hope others with specific experience on primary glia cell stimulation experiments can provide more input.  
Best,
Ricardo
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To improve data and reduce variability i would like to move away from outbred strain (Sprague Dawley) and use inbred rats. But it is nearly impossibly to work out which strain to use from the available ones: Lewis, Long Evans, Fischer, Kyoto and many more. I know, for example, that Lewis rats are used for some neuroimmunology (EAE model) but this is different to our model (effect of systemic LPS on the brain) and therefore might not be directly relevant. Do people have any experience with this type of work and inbred rats?
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Dear Diana,
As mentioned above i recommend the dark agouti rats. They are an invred strain that are great for reliable EAE research as immunisation with IFA and recombinant MOG is sufficient, you dont require further addition of pertussis or other complete adjuvants. 
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The process of myelinogenesis starts at birth and last until 22 years of age.
Sleep may play an important role in this process.
In demyelinising diseases such MS sleep disturbances are the rule.
Perhaps it will be interesting for this studies the myelin mutant taiep rat model
by José R. Eguibar, Instituto de Fisiología, Universidad Autónoma de Puebla. Mexico)
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Hi Marco,
Thanks for your reference that I did not know.
Best,
Rosa
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Neurotoxin, Cuprizone, is used for demyelination in mouse brain for de and remyelination. My inquiry is how it demyelinates?  
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Hi Monokesh,
Cuprizome is a copper chelator and subsequent copper deficit alters mitochondrial function in the brain and induces disturbance of energy metabolism in oligodendroglia leading to apoptosis. The selective vulnerability of oligodendroglia has been linked to myelin synthesis which requires vast amounts of energy.
You might also like having a look at this review article:
Cuprizone-induced demyelination as a tool to study remyelination and axonal protection http://www.ncbi.nlm.nih.gov/pubmed/23666824
Hope this helps. Best, Norbert
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Humoral response has a profound effect on M.S. symptoms
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My group (Kleinstein lab) has done some work; a recent publication is attached
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We are finding a significant role of neuro invasive viruses in the instigation and promotion of the Multiple Sclerosis. Is there any other scientist out there who has already conducted some research work on this subject?
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 Dear Dr. Dieter Huisman,
Thank you for the links to your references. Most of them are unsafe files, Sorry I couldn't read them. But I did have a chance to look at the BCA -Clinic, what a wonderful service. I would like to speak to you and Dr. Nicolaus about our research, as to how we can compliment your institution with the "Multi-Systemic Disease.
Best Regards
Prof. Dr. Shahid Sheikh
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I require a mRNA marker for microlgia for qPCR analysis and was wondering if IFNg would be a suitable candidate.  I am looking at microglia in quail and commerical gene sequences are hard to come by but I have one for IFNg.  I undertsand IFNg activates microglia so would this be a viable candidate?#
Thanks
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Dear David as it was told by other friends, your marker is not so appropriate, currently i'm working on EAE model, and i've searched about microglia, eventually i understood that CD1125, CD45, Cx3cr1, Iba1, CD68 would be best marker. i suggest you to use IHC technique for assessing microglia activity. the article link is attached for you. its can solve your problem. maybe
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I've been trying to culture human fetal microglia from whole human fetal brain samples.  I've been using the protocol by Durafort, et al. (link attached).  Of course the first time I tried the protocol it worked great, but every sample thereafter has been resulting in nearly complete cell death.  
The first time I did the prep there were clearly lots of cells that stuck to the flask the next day and my total microglia yield was on target for what the authors reported.  Now the cells that are there barely stick to the flask and are easily lifted just by gently moving the flask, and they look nothing like the cells that I got the first time I completed the prep.
The one difference is that the first time I used heat-inactivated FBS and since then I've been using charcoal-stripped FBS.  Otherwise the media is the same (DMEM, 5% FBS, 1% pen/strep).
As I've only tried the prep a couple of times, I'm wondering if this is something with sample variability or maybe even if there is something going wrong in how the samples are being shipped to us (in media vs. PBS, in which case the cells may just be dying in the PBS).  I just completed a prep yesterday and will have another look in a few days (I don't want to touch the flasks just in case any cells may get lifted).
If you happen to know of a method that works or tips or tricks that work for them I would really appreciate it!  If you have any questions for me let me know.  Thanks!
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Is this tutorial video helpful for you? The procedure is shown for three adult mouse brains but it is the same for human brain tissue.
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Macrophages are known to be an active player in MS. They are involved in active demyelination and myeline uptake, which leads towards the development of foamy macrophages with M2-like properties. But can they migrate out of the brain/lesion before they become necrotic and cause a pro-inflammatory respons (cfr. atherosclerosis)?
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Foamy macrophages (gitter cells) do migrate out of the MS plaque into the perivascular space and then into the blood stream.
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I am trying to prepare an anisomycin solution for injection into the brains of my rats. I am dissolving the ANI in equimolar HCl ( 1 mmol of ANI for 1mmol of HCl). The ANI dissolves easily in HCl. I then diluted the HCl a bit with saline, which was also no problem, but when I adjusted the pH of my solution with NaOH the ANI fell out of solution again. 
Any suggestion? 
Thanks
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Thanks for your suggestions. 
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At present I am member of the Laboratory of Clinical Neuroimmunology in Institut de Recerca del Hospital Universitari Vall d'Hebron (Barcelona, Spain). We are concerned with Multiple Sclerosis research and now we are isolating Treg cells from human blood. I have read researchers to obtain about million cd4+cd25+cd127lo/- cells from 20 mL blood sample. After several attempts, we get an average of half a million cells from 64 mL blood sample. We use the CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II (MACS, from Miltenyi Biotec). We use either one LD column and two MS columns or two LD columns and one MS column. Could I know which protocol do you follow? Thanks.
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Please refer to Methods in the following articles
1. Clin Exp Immunol. 2013 Aug;173(2):310-22.
Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials.
Berglund D, et al.
Adoptive transfer of regulatory T cells (T(regs)) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with T(reg) therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4(+) CD25(high) CD127(low) T(regs) from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare T(reg) preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded T(reg) preparations have been characterized by their purity, cytokine production and in-vitro suppressive ability. The results show that T(reg) preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the T(reg) preparations. In particular, FACS-sorted T(reg) preparations expanded with mature DCs secrete more interleukin (IL)-10 and granzyme B than FACS-sorted T(reg) preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with T(regs) from uraemic patients that may guide future efforts to produce clinical-grade T(regs) for use in kidney transplantation.
2. Am J Respir Crit Care Med. 2013 Jun 1;187(11):1249-58.
Regulatory T cells attenuate mycobacterial stasis in alveolar and blood-derived macrophages from patients with tuberculosis.
Semple PL(1), Binder AB, Davids M, Maredza A, van Zyl-Smit RN, Dheda K.
RATIONALE: There are hardly any data about the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (T-Regs) in the lungs of patients with active tuberculosis (TB). OBJECTIVES: To obtain data about the frequency of CD4(+)CD25(+)Foxp3(+) T-Regs, and their impact on mycobacterial containment, in the lungs of patients with active TB. METHODS: Patients with pulmonary TB (n = 49) and healthy volunteers with presumed latent TB infection (LTBI; n = 38) donated blood and/or bronchoalveolar lavage (BAL) cells obtained by bronchoscopy. T-cell phenotype (Th1/Th2/Th17/T-Reg) and functional status was evaluated using flow-cytometry and (3)H-thymidine proliferation assays, respectively. H37Rv-infected alveolar and monocyte-derived macrophages were cocultured with autologous T-Regs and purified protein derivative (PPD) preprimed T-Reg-depleted effector cells. Mycobacterial containment was evaluated by counting CFUs. MEASUREMENTS AND MAIN RESULTS: In blood and BAL T-Reg levels were higher in TB versus LTBI (P < 0.04), and in TB the frequency of T-Regs was significantly higher in BAL versus blood (P < 0.001). T-Reg-mediated suppression of T-cell proliferation in blood and BAL was concentration-dependent. Restriction of mycobacterial growth in infected alveolar and monocyte-derived macrophages was significantly diminished, and by up to 50%, when T-Regs were cocultured with PPD-primed CD4(+) effector T cells. The levels of CD8(+) T-Regs (CD8(+)CD25(+)Foxp3(+)), IL-17-producing T-Regs (IL-17(+)CD4(+)CD25(+)Foxp3(+)), and IL-17-producing T cells were similar in BAL-TB versus BAL-LTBI. Within the TB group compartmentalization of responses was prominent (T-Reg, IFN-γ, tumor necrosis factor-α, IL-17, and IL-22 significantly higher in BAL vs. blood). CONCLUSIONS: In patients with TB the alveolar compartment is enriched for CD4(+) T-Regs. Peripheral blood-derived T-Regs decrease the ability of alveolar and monocyte-derived macrophages to restrict the growth of mycobacterium tuberculosis in the presence of effector cells. Collectively, these data suggest that CD4(+)CD25(+)FoxP3(+) T-Regs subvert antimycobacterial immunity in human TB.
3. Pathol Res Pract. 2011 Apr 15;207(4):209-15.
T(reg) cells: collection, processing, storage and clinical use.
Daniele N, et al.
T regulatory cells are fundamental in the maintenance of immune homeostasis and self-tolerance. Experimental models suggest the existence of two functional types of T(reg) cells designated naturally occurring and induced. Interest in T(reg) cells increased with evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in the treatment of various pathological conditions. The existence of a subpopulation of suppressive T cells was the subject of significant controversy among immunologists for many years. T regulatory cells limit immune activation through a variety of direct and indirect interactions, many of which are yet to be determined. Fully understanding T(reg) cells biology will lead us to harnessing the capacity of these cells in order to develop strategies to prevent autoimmune disorders and tolerance to transplantation. Efficient isolation, expansion and cryopreservation strategies that comply with Good Manufacturing Practice (GMP) guidelines are prerequisites for the clinical application of human CD4+ CD25+ CD127(low) FOXP3+ regulatory T cells.
4. Rheumatology (Oxford). 2011 Apr;50(4):646-56.
FoxP3+ CD4+ T cells in systemic autoimmune diseases: the delicate balance between true regulatory T cells and effector Th-17 cells.
Abdulahad WH, Boots AM, Kallenberg CG.
Breakdown of tolerance is a hallmark of autoimmune diseases. Over the past 10 years, there has been increased interest in the role of FoxP3(+) regulatory T cells (T(Regs)) in maintaining peripheral tolerance. Dysfunction of these cells is considered to play a major role in the development of autoimmune diseases. Besides their suppressive function, a fraction of these cells has the capacity to differentiate into IL-17-producing cells (Th-17), a phenomenon associated with autoimmune inflammation. The revealed plasticity of T(Regs), therefore, has obvious implications when designing therapeutic strategies for restoring tolerance in autoimmune diseases using T(Regs). In this review, we discuss development, classification, molecular characterization and mechanisms of suppression by T(Regs). In addition, we describe recent data on their potential conversion into Th-17 cells in human systemic autoimmune diseases. We also outline a new strategy for T(Reg)-based therapy via isolation, expansion and re-infusion of highly pure FoxP3(+) T(Regs) free of contaminating effector T cells.
5. Clin Exp Immunol. 2009 May;156(2):246-53.
Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications.
Di Ianni M(1), et al.
Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.
6. J Exp Med. 2007 Sep 3;204(9):2159-69.
Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis.
Scott-Browne JP(1), Shafiani S, Tucker-Heard G, Ishida-Tsubota K, Fontenot JD,
Rudensky AY, Bevan MJ, Urdahl KB.
Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4(+) T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4(+) T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed approximately 1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.
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transport of rabies virus in neurons
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please review the following:
Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery. PLoS Pathog 10(8): e1004348
Shani Gluska, et al.
Abstract
Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV-G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS.
Rabies virus as a transneuronal tracer of neuronal connections. Adv Virus Res. 2011;79:165-202.
Ugolini G1.
.
Abstract
Powerful transneuronal tracing technologies exploit the ability of some neurotropic viruses to travel across neuronal pathways and to function as self-amplifying markers. Rabies virus is the only viral tracer that is entirely specific, as it propagates exclusively between connected neurons by strictly unidirectional (retrograde) transneuronal transfer, allowing for the stepwise identification of neuronal connections of progressively higher order. Transneuronal tracing studies in primates and rodent models prior to the development of clinical disease have provided valuable information on rabies pathogenesis. We have shown that rabies virus propagation occurs at chemical synapses but not via gap junctions or cell-to-cell spread. Infected neurons remain viable, as they can express their neurotransmitters and cotransport other tracers. Axonal transport occurs at high speed, and all populations of the same synaptic order are infected simultaneously regardless of their neurotransmitters, synaptic strength, and distance, showing that rabies virus receptors are ubiquitously distributed within the CNS. Conversely, in the peripheral nervous system, rabies virus receptors are present only on motor endplates and motor axons, since uptake and transneuronal transmission to the CNS occur exclusively via the motor route, while sensory and autonomic endings are not infected. Infection of sensory and autonomic ganglia requires longer incubation times, as it reflects centrifugal propagation from the CNS to the periphery, via polysynaptic connections from sensory and autonomic neurons to the initially infected motoneurons. Virus is recovered from end organs only after the development of rabies because anterograde spread to end organs is likely mediated by passive diffusion, rather than active transport mechanisms.
Rabies Virus Envelope Glycoprotein Targets Lentiviral Vectors to the Axonal Retrograde Pathway in Motor Neurons. 2014 The Journal of Biological Chemistry, 289, 16148-16163.                                    
James N. Hislop, et al.
Abstract
Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.
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I am considering using a scholl analysis (using imagej software) but I was wondering if anyone has any better ideas.  I only need to determine if the microglia are activated, I don't need to describe their morphology in detail.  
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If you also have IBA-1 stained control sample then you can compare IBA-1 staining intensity between them. Activated microglia show increased IBA-1 staining. You can also quantify staining intensity in ImageJ. This is one of the simplest ways.
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Could you suggest any good marker and corresponding antibody you employed to mark synapses? PSD95 seems a suitable option. I am using it on primary neuron culture from mouse cortex and hippocampus, but if it would work on cerebellar cultures would be also fine.
Thanks
francesco
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I concur with Synaptophysin (>90-95% localisation on synaptic vesicles) as a good marker for the general presynaptic compartment and with Bassoon as a marker for the active zone.
Personally, I've had very good experience with the Synaptic Systems #101004 guinea pig antibody against Synaptophysin (http://sysy.com/products/s-physin1/facts-101004.php) and I find the Enzo SAP7F407 mouse monoclonal against Bassoon (http://www.enzolifesciences.com/ADI-VAM-PS003/bassoon-mab-sap7f407/) outstanding. Both antibodies are particularly good in ICC and IHC.
Good markers for the postsynapse include PSD-95 and Homer1.
The Synaptic Systems #160003 rabbit anti Homer1 (http://sysy.com/products/homer1/facts-160003.php) seems to be very good and colleagues of mine had good experience with PSD-95 antibodies from Cell Signaling.
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I'm looking for an IHC or mRNA marker that specifically identifies ramified (unactivated) microglia so I can distinguish between an inactive and active phenotype.  Can anyone help please?  Thanks
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Dear David,
All antibodies against microglial cells can  provide the images where  you can figure out activated or in inactivated state of these cells.  Marker Iba1 is the best, which is upregulated during activation. Good luck. Tatyana
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I'd like to perform an experiment on mice inducing EAE with whole myelin as the immunogen. The most important reference seems to be "Oct;21(4):749-57.
Myelination in rat brain: method of myelin isolation. Norton WT, Poduslo SE.J. of Neurochemistry 1973" that obviously is about rats.. Thank you in advance for your help.
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Just a few simple pratical suggestions.
For an optimal immunization animals must receive an intradermal injection of the antigen in a depot form that will release it slowly. For this purpose any lyophilized preparation must be reconstituted in water or in saline solution up to the appropriate concentration and the suspension must be emusioned with Complete or Incomplete Freund's Adjuvant until a thick compound similar to a cream will be obtained. This is usually carried out connecting two opposed syringes with a two/three way connector one containing  the Freund's adjuvant and one the water/saline suspension (a connector can also be built in house, connecting a  large caliber needle with the cap of a secon identical needle). Pumping grdually the water solution in the oil a nice thick emulsion will be easily obtained. The thickness of the emulsion is critical for a efficacy of the depot.
Animals can be injected in the foot paths or in the skin around shoulder or hind limb joints, with  insulin syringe and needle. There isn't much room for large volumes of emulsion in these sites, therefore the concentration of the antigen in the emulsion must be carefully planned in advance.
Purification of Myelin from brain or spinal cord is simple, all the methods are based on gradient centriugations. In addition I believe that at least bovine and mouse purified  myelin prparations can be purchased from the market.
However, as already suggested by others, fresh brain white matter or spinal cord, whatever the source, will work nicely as well. In this case fresh tissue can be lyophilized and stored or can be used as a fresh preparation. If used fresh it may not be necessary to dissoleve it in saline solution
Best regards, and good luck with your experiments.
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Recently I checked the acsf pH before and after 95% O2/ 5% CO2 buffering and found pH value is 7.8 (7.8) and 7.6 (after) with 26mM NaHCO3 and 5% CO2. Does anyone know how to calculate the buffer ability of CO2-NaHCO3 pair? And what's the pH range of your acsf before and after buffering?
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The pH can be adjusted with HCl before carbogenation to ensure you are starting in the physiological range.  If the pH is still high after carbogenation, try lowering NaHCO3 from 26mM to 24mM.  That is what I use.  Alternately, you can get better buffering by using a formulation with HEPES and NaHCO3.  A small amount of HEPES will lower the pH.  For example, you can add 5 mM HEPES to your existing formula.
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Dear all, I am Rajaram.
At present I am doing depression studies on animals. I also want to check the same in vitro on cell lines. If any cell lines are there (like BV-2 microglia) please suggest some cell lines. Which is the ideal cell line for checking the LPS-induced depressive like behavior in vitro?
Thanks in advance..
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thanks a lot mam...
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I am interested to know why C6 is the famous cell line to be chosen to co-culture with bEnd.3 (endothelial cells). 
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Hello Robert. Thank you for your answer. However to answer your question on why bEnd.3 is chosen, it is largely due to personal interest. It is then supported by the few articles especially by Brown et al. 2007 which claimed that bEnd.3 has rapid growth, maintains BBB characteristics over repeated passages, forms the functional barriers, and amenable to molecular interventions. 
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k
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Dear Saroj Please find paper where you can get  protocol for the GSH, MDA in rat brain tissue. PDF is attached here.
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I am trying to induce passive EAE by adoptive transfer of primed T cells from WT C57BL/6 donors to WT C57BL/6 recipients. The protocol I followed was:
Day 0 - immunised donor mice with 200ug MOG35-55 s.c. + one does of 200ng pertussis toxin i.p.
D 8 - sacrifice donors and remove lymph nodes and put cells in culture with 20ug/ml MOG + 10ng/ml IL-23
Day 11 - blasting cells observed. Transferred 10 million cells i.p. per sub-lethally irradiated WT recipient (irradiated approximately 3 hours prior to cell injection). =D0 of scoring EAE.
Now it is Day 15 post cell transfer and my mice are not displaying any EAE symptoms. I see some protocols also give pertussis with the cell injection into recipients and again two days later? Or did I inject too few cells? What could the problem be?
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That's because Th2 polarized cells (IL23 stimulated) do not induce EAE w/ MOG peptide! And if they do in the sub lethal irr mice  it will be a milder form, with limited clinical manifestation. The only reference I've seen for Th2 pol. cells inducing EAE is with MBP, not MOG,  and the clinical symptoms take longer to develop.
Below is a procedure we routinely use, with robust disease.
Procedure:
day 0:
Mice are anesthetized and injected subcutaneously with a total of 200μL over two areas 
(between shoulder blades, above right hip) with 3 mg/ml MOG35-55
 mixed 1:1 with 3 mg/ml Mycobacterium tuberculosis in Incomplete Freund's Adjuvant on day 0.
(300 μg peptide and 300 μg Mycobacterium tuberculosis [H37Ra])
Two hours later, mice are injected intraperitoneally with 100 μL of 300 ng Pertussis Toxin.
day 2:
Mice will be injected intraperitoneally with 100 μL of 300 ng Pertussis Toxin.
day 9:
Draining LN (DLN) cells are collected
Polarization of lymph node cells
day 0: (7 days after culture)
Prep of  polarized cells.
Cells adoptly transferred i.v. into naive C57Bl/6 mice @ 15x106 cells/ms
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I am searching for human microglial cell lines (best would be immortalized and easily transfectable) and I would really appreciate any hint or comment.
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Thanks Ian, I received them from a lab in Canada, unfortunately these cells did not have certain proteins expressed on microglial cells, and after cell authentification, the company told me that my cells are of rat origin with no signal for human! I will try another batch from a different lab but I'm quite skeptical ;)
cheers
Fargol
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And neurodegeneration in mice.
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There are multiple ways to assess neuroinflammation. You can perform immunohistochemistry by using specific markers for microglial activation (Iba1) , astrocytic response (GFAP), and neuronal loss (NeuN or Fluorojade for dying neurons). You can also look at the levels of various cytokines that are upregulated during inflammation (TNF-alpha, Interleukins, TGF-beta, and so on).
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Tau oligomers and aggregates have been shown to be the major target, on the basis of our neuroimmunomodulation theory of Alzheimer´s pathogenesis.
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Our group has pioneering shed light on important aspects of the mechanisms underlying the adverse events occurring during immunization trials for AD, demonstrating for the first time that anti-Aβ antibody and the transient manifestation of these events (called Amyloid-related Imaging Abnormalities - ARIA-) are linked to a rapid removal of the deposited cerebrovascular Aβ, strictly modulated by antibody concentration. In agreement with AD immunization, we also observed a reduction of both antibodies and neurodegenerative markers (tau, P-tau, Aβ40 and Aβ42) after clinical and radiological remission, either spontaneously or after treatment.
These findings were collected in a rare disease named Cerebral amyloid angiopathy–related inflammation (CAA-ri), a meningoencephalitis affecting older adults and characterized by rapidly progressive cognitive decline, seizures, headaches, T2-hyperintense MRI lesions, FLAIR vasogenic edema, and neuropathologic evidence of CAA-associated vascular inflammation. Recently, CAA-ri has generated enormous interest in the field of Alzheimer’s disease (AD) for its clinical and radiological similarities to the ARIA developed by a subset of AD patients during experimental immunization trials with antibody to amyloid beta (Aβ) and/or secretase inhibitors.
These aspects are timely and could provide an important challenge for the field, leading to critical information for the ongoing clinical trials. If confirmed in a large number of patients, such as we are doing through the iCAB International Network, the possibility to follow up the CSF levels of anti-Aβ antibody would allow maintaining a putative “therapeutic window” for the safe clearance of Aβ from the brain, limiting the occurrence of CAA-related consequences and placing anti-Aβ antibodies as a promising therapy not only for AD but also for CAA.
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A-804598 is an antagonist for P2X7 receptors. We're trying to find out if there's an in vivo data in relation to toxicity for this particular antagonist. Please let me know if there's anything, much appreciated.
- It's an 8 weeks chronic mild stress mice model, interest being the polarization of microglia/macrophages and NLRP3 inflammasome activation in CNS. Administration route is via gavaging syringe. (updated 08/04/2014)
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Is a radioligand important? If not, you could use Brillian Blue G (IV) as a selective P2X7 blocker and then do immunohisto for analysis. BBG is very similar to blue food coloring so it is rather safe. Sorry I don't know more about your specific compound. All the best in your research.
Jiang L-H, Mackenzie AB, North RA, Surprenant A. Brilliant Blue G Selectively Blocks ATP-Gated Rat P2X7 Receptors. Mol Pharmacol 2000; 58: 82–8.
Peng W, Cotrina ML, Han X, Yu H, Bekar L, Blum L, et al. Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. PNAS 2009; 106: 12489–93.
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.
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Dear Sir, thank You for the answer. I have tried gabapentin, but it causes me all day sleepy, so I was unable to function normally ;) At this moment I get episcleritis with exfoliative cheilitis and because of some problems with gut, it is higly possibly, that it might be a collitis ulcerosa which is the basis of all problems. The colonoscopy will reaveal the situation and will see. The blood results are ok. But they are always ok, even when the flare is present. Best regards. Alex
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I still don't know what kind of injection I am going to perform (intraperitoneal or intracerebral) to obtain the best staining.´Does anyone has experience in what could be the best way of injection/timing of LPS? Thanks!
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Simone, could you let us know what the aim of your experiment is as they will determine the best advice to provide? LPS by either route will certainly elicit a robust Iba; within an hour for ICV and 4 hours for IP.
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I am looking for protocols to develop a co-culture system and then introduce a virus in the culture so that the macrophage can present it to the T-cells.
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yes! could you email it to me at priya543@gmail.com Thank you!
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We need to identify neurons in the brainstem, but cannot use NeuN made in mouse, which is the most frequently used, because our other antibodies are made in mouse, and I cannot find in the literature any experimental work with rabbit NeuN. Has anybody used any commercial one? thanks!!
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NeuN is not a pan-neuronal marker. It is chiefly a marker of granule cells (and then only labels a fraction of them, and this proportion is sensitive to neurodegenerative insult, see PMID: 15223381). If you are looking for a pan-neuronal marker you would be better served with neuron specific enolase, tuj1 or HuC.
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I am at the annual BD nordic meeting, where the possibility of BD producing fluorescently labelled anti-mouse dopamine receptors antibodies suitable for flow cytometry has come up if there is enough interest in the scientific community. Anybody interested? Which subtypes would be the most interesting?
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Dopamie receptors are situated mainly in the Substantia Nigra(SN) in the basis of the brain. These receptors i.e. their function is very important in neuroimmunology and especialy in autoimmune reactions in the brain. The most important functions of these receptors is in the development of Parkisonş disease. D2 receptors are especialy interesting from psychological aspect, while D1 preferentially for motor functions of the brain. I believe that in the periphery nerve system they are also interesting not only in flow cytometry but also for confocal microscopy and atomic microscopy as well.
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I am working on a connection with colic and brain development in infants.
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Thank you! I am investigating the connection with early dysregulation symptoms in infants (colic) and later development of Sensory Processing Disorder in children. I am looking at the microbiome's possible influence in this neurodevelopmental disorder. As you may know, rodent studies are finding a disturbed HPA axis in GF mice with a critical window of development for a healthy stress response. I wonder if this is also happening in a sub-set of infants ( those who do not regulate at 3 months per Wessel's criteria).
Thank you so much for your response and support!! My daughter has SPD and I need to find answers to help our family and others like us.
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Mice are perfused with saline and 4% PFA, followed by 4hr fixation in 4% PFA. Brains are then placed in 30% sucrose overnight, then sectioned at 30um on a freezing microtome. Sections are then placed on slides, dried and washed with PBS. I then utilized a variety of different antigen retrieval steps, blocking buffers, and antibody dilutions. The following protocols are what I've tried so far:
Wash (3x PBS)
Block for 60 min with 4% NGS in 1% BSA, 0.4% TX-100 PBS
Incubate in 1° Ab (1:1k with Anti-Iba1 from WAKO, Ab is for IHC) overnight at 4C
Wash
Peroxidase removal with 1% H2O2 for 15min
Wash
Incubate in 2° ab (Goat Anti-rabbit) in BSA/TX/PBS
Wash
ABC incubation
Wash
DAB
This protocol results in very spotty staining pattern, various regions of cortex stain, nothing in the striatum or hypothalamus. I've tried both free floating and staining on slides. Where the staining does work it works beautifully, I see complete microglia morphology and no background stain. However this only occurs in ~5% of any tissue section (not 5% of samples, but 5% of a tissue slice).
So I tried a variety of different antigen retrieval steps including
50 mM TBS + 0.05% Tween 20, pH 9.0 for 20 min at 99°C
10mM TE + 0.05% Tween 20, pH 9.0 for 20 min at 99°C
10mM NA citrate buffer + 0.05% Tween 20, pH 6.0 for 30 min at 80°C
Tissue was then blocked with 10% Fetal Bovine Serum, 3% BSA, tx-PBS for 60min then incubated ON in Anti-Iba1 (1:2.5k, WAKO)
Then I followed the above protocol
This resulted in mixed but complete staining.
The tissue incubated in TBS had very weak, but uniform Iba staining (only cell bodies stained, no processes).
Tissue incubated in TE showed a stronger uniform Iba staining but again only cell bodies stained.
Tissue incubated in SSC showed the best staining pattern, I now see some processes, but its still far from how surveying microglia should appear, plus I now have massive background staining.
So I tried the SSC antigen retrieval method again, this time I blocked with 4% NGS, BSA/Tx/PBS, and followed the above protocol. This results in the worst staining I've yet to see, massive background only cell bodies stain.
Any thoughts, tips, tricks, etc for staining Iba-1 in mouse brain are very welcome!
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I would like to reiterate what Gloria has stated: Iba1 staining in mouse or rat brain does not need all those pretreatments, only borohydride to destroy aldehyde and Schiff bases and Triton X-100 for permeabilization. I use Wako Iba1 at 1:40,000 preferring to dilute the Vector biotinylated secondary antibody and ABC reagent to reduce costs with large volume free floating sections, see attached image.
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The protocol is working very well in Polyethylene glycol embedded tissue. All of the sections are labelled on slides. We have to use 6u sections so that we can get sufficient cell counts with BrdU/NeuN. However, we are trying to process these same sections for numbers of activated and ramified microglial numbers in the hippocampus. I am having difficulties determining how to analyze the morphology of the ramified microglial cells, as their cell bodies are very small and their processes are extending into other sections of tissue. Any ideas?
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Dear Kimberly,
We have done a couple of studies specifically looking at changes in microglial morphology.
There are a lot of ways you can get at this information. One of simplest is to use a freeware program like imagej or Fiji. Using these packages you can convert your raw image (say a .tiff or .jpg) into a binary image and then using this new image assess basic metrics such as max diameter, length roundness etc. This is pretty good place to start. There are some other interesting packages - like fraclac which you can use to get into issues like how fractal the shape of the cells are.
If you want to go a step further then some commercial packages may be of benefit. We have extensively used Neurolucida from MBF biosciences. Using this when coupled with automated XYZ stage it is relatively straightforward to create high fidelity reconstructions of microglia at 100x. We tend to find depending on their complexity it takes 10-30minutes/cell to reconstruct. We then port the digital reconstruction's into the companion product NeuroExplorer which you can use to produce an almost limitless list of measurements. Of these I particularly like Scholl analysis undertaken on branch length, branch number and volume.
We have recently started working with Imaris and this is really nice particularly for semi-automated 3D reconstructions. In essence, however, it produces data similar to Neurolucida.
Best
Rohan.